Risk of Gastroenteritis from Swimming at a Wastewater-Impacted Tropical Beach Varies across Localized Scales.

Population growth and changing climate are expected to increase human exposure to pathogens in tropical coastal waters. We examined microbiological water quality in three rivers within 2.3 km of each other that impact a Costa Rican beach and in the ocean outside their plumes during the rainy and dry seasons. We performed quantitative microbial risk assessment (QMRA) to predict the risk of gastroenteritis associated with swimming and the amount of pathogen reduction needed to achieve safe conditions. Recreational water quality criteria based on enterococci were exceeded in >90% of river samples but in only 13% of ocean samples. Multivariate analysis grouped microbial observations by subwatershed and season in river samples but only by subwatershed in the ocean. The modeled median risk from all pathogens in river samples was between 0.345 and 0.577, 10-fold above the U.S. Environmental Protection Agency (U.S. EPA) benchmark of 0.036 (36 illnesses/1,000 swimmers). Norovirus genogroup I (NoVGI) contributed most to risk, but adenoviruses raised risk above the threshold in the two most urban subwatersheds. The risk was greater in the dry compared to the rainy season, due largely to the greater frequency of NoVGI detection (100% versus 41%). Viral log10 reduction needed to ensure safe swimming conditions varied by subwatershed and season and was greatest in the dry season (3.8 to 4.1 dry; 2.7 to 3.2 rainy). QMRA that accounts for seasonal and local variability of water quality contributes to understanding the complex influences of hydrology, land use, and environment on human health risk in tropical coastal areas and can contribute to improved beach management. IMPORTANCE This holistic investigation of sanitary water quality at a Costa Rican beach assessed microbial source tracking (MST) marker genes, pathogens, and indicators of sewage. Such studies are still rare in tropical climates. Quantitative microbial risk assessment (QMRA) found that rivers impacting the beach consistently exceeded the U.S. EPA risk threshold for gastroenteritis of 36/1,000 swimmers. The study improves upon many QMRA studies by measuring specific pathogens, rather than relying on surrogates (indicator organisms or MST markers) or estimating pathogen concentrations from the literature. By analyzing microbial levels and estimating the risk of gastrointestinal illness in each river, we were able to discern differences in pathogen levels and human health risks even though all rivers were highly polluted by wastewater and were located less than 2.5 km from one another. This variability on a localized scale has not, to our knowledge, previously been demonstrated.

Relationships among microbial indicators of fecal pollution, microbial source tracking markers, and pathogens in Costa Rican coastal waters.

Tropical coastal waters are understudied, despite their ecological and economic importance. They also reflect projected climate change scenarios for other climate zones, e.g., increased rainfall and water temperatures. We conducted an exploratory microbial water quality study at a tropical beach influenced by sewage-contaminated rivers, and tested the hypothesis that fecal microorganisms (fecal coliforms, enterococci, Clostridium perfringens, somatic and male-specific coliphages, pepper mild mottle virus (PMMoV), Bacteroides HF183, norovirus genogroup I (NoVGI), Salmonella, Cryptosporidium and Giardia) would vary by season and tidal stage. Most microorganisms' concentrations were greater in the rainy season; however, NoVGI was only detected in the dry season and Cryptosporidium was the only pathogen most frequently detected in rainy season. Fecal indicator bacteria (FIB) levels exceeded recreational water quality criteria standards in >85% of river samples and in <50% of ocean samples, regardless of the FIB or regulatory criterion. Chronic sewage contamination was demonstrated by detection of HF183 and PMMoV in 100% of river samples, and in >89% of ocean samples. Giardia, Cryptosporidium, Salmonella, and NoVGI were frequently detected in rivers (39%, 39%, 26%, and 39% of samples, respectively), but infrequently in ocean water, particularly during the dry season. Multivariate analysis showed that C. perfringens, somatic coliphage, male-specific coliphage, and PMMoV were the subset of indicators that maximized the correlation with pathogens in the rivers. In the ocean, the best subset of indicators was enterococci, male-specific coliphage, and PMMoV. We also executed redudancy analyses on environmental parameters and microorganim concentrations, and found that rainfall best predicted microbial concentrations. The seasonal interplay of rainfall and pathogen prevalence undoubtedly influences beach users' health risks. Relationships are likely to be complex, with some risk factors increasing and others decreasing each season. Future use of multivariate approaches to better understand linkages among environmental conditions, microbial predictors (fecal indicators and MST markers), and pathogens will improve prediction of high-risk scenarios at recreational beaches.

Quantification of Protozoa and Viruses from Small Water Volumes.

Large sample volumes are traditionally required for the analysis of waterborne pathogens. The need for large volumes greatly limits the number of samples that can be processed. The aims of this study were to compare extraction and detection procedures for quantifying protozoan parasites and viruses from small volumes of marine water. The intent was to evaluate a logistically simpler method of sample collection and processing that would facilitate direct pathogen measures as part of routine monitoring programs. Samples were collected simultaneously using a bilayer device with protozoa capture by size (top filter) and viruses capture by charge (bottom filter). Protozoan detection technologies utilized for recovery of Cryptosporidium spp. and Giardia spp. were qPCR and the more traditional immunomagnetic separation-IFA-microscopy, while virus (poliovirus) detection was based upon qPCR versus plaque assay. Filters were eluted using reagents consistent with the downstream detection technologies. Results showed higher mean recoveries using traditional detection methods over qPCR for Cryptosporidium (91% vs. 45%) and poliovirus (67% vs. 55%) whereas for Giardia the qPCR-based methods were characterized by higher mean recoveries (41% vs. 28%). Overall mean recoveries are considered high for all detection technologies. Results suggest that simultaneous filtration may be suitable for isolating different classes of pathogens from small marine water volumes. More research is needed to evaluate the suitability of this method for detecting pathogens at low ambient concentration levels.

Assessment of sources of human pathogens and fecal contamination in a Florida freshwater lake.

We investigated the potential for a variety of environmental reservoirs to harbor or contribute fecal indicator bacteria (FIB), DNA markers of human fecal contamination, and human pathogens to a freshwater lake. We hypothesized that submerged aquatic vegetation (SAV), sediments, and stormwater act as reservoirs and/or provide inputs of FIB and human pathogens to this inland water. Analysis included microbial source tracking (MST) markers of sewage contamination (Enterococcus faecium esp gene, human-associated Bacteroides HF183, and human polyomaviruses), pathogens (Salmonella, Cryptosporidium, Giardia, and enteric viruses), and FIB (fecal coliforms, Escherichia coli, and enterococci). Bayesian analysis was used to assess relationships among microbial and physicochemical variables. FIB in the water were correlated with concentrations in SAV and sediment. Furthermore, the correlation of antecedent rainfall and major rain events with FIB concentrations and detection of human markers and pathogens points toward multiple reservoirs for microbial contaminants in this system. Although pathogens and human-source markers were detected in 55% and 21% of samples, respectively, markers rarely coincided with pathogen detection. Bayesian analysis revealed that low concentrations (<45 CFU × 100 ml(-1)) of fecal coliforms were associated with 93% probability that pathogens would not be detected; furthermore the Bayes net model showed associations between elevated temperature and rainfall with fecal coliform and enterococci concentrations, but not E. coli. These data indicate that many under-studied matrices (e.g. SAV, sediment, stormwater) are important reservoirs for FIB and potentially human pathogens and demonstrate the usefulness of Bayes net analysis for water quality assessment.

Daily measures of microbes and human health at a non-point source marine beach.

Studies evaluating the relationship between microbes and human health at non-point source beaches are necessary for establishing criteria which would protect public health while minimizing economic burdens. The objective of this study was to evaluate water quality and daily cumulative health effects (gastrointestinal, skin, and respiratory illnesses) for bathers at a non-point source subtropical marine recreational beach in order to better understand the inter-relationships between these factors and hence improve monitoring and pollution prevention techniques. Daily composite samples were collected, during the Oceans and Human Health Beach Exposure Assessment and Characterization Health Epidemiologic Study conducted in Miami (Florida, USA) at a non-point source beach, and analyzed for several pathogens, microbial source tracking markers, indicator microbes, and environmental parameters. Analysis demonstrated that rainfall and tide were more influential, when compared to other environmental factors and source tracking markers, in determining the presence of both indicator microbes and pathogens. Antecedent rainfall and F+ coliphage detection in water should be further assessed to confirm their possible association with skin and gastrointestinal (GI) illness outcomes, respectively. The results of this research illustrate the potential complexity of beach systems characterized by non-point sources, and how more novel and comprehensive approaches are needed to assess beach water quality for the purpose of protecting bather health.

Presence of pathogens and indicator microbes at a non-point source subtropical recreational marine beach.

Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, enterococci, and Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp., and Giardia spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium V. vulnificus in sand and water, detection of the allochthonous protozoans Giardia spp. in water, and detection of Cryptosporidium spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.

Quantification of human polyomaviruses JC Virus and BK Virus by TaqMan quantitative PCR and comparison to other water quality indicators in water and fecal samples.

In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify > or =10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35 degrees C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens.

Detection of human-derived fecal pollution in environmental waters by use of a PCR-based human polyomavirus assay.

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 microl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking "toolbox."

Microbial source tracking by DNA sequence analysis of the Escherichia coli malate dehydrogenase gene.

Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.

Validity of the indicator organism paradigm for pathogen reduction in reclaimed water and public health protection.

The validity of using indicator organisms (total and fecal coliforms, enterococci, Clostridium perfringens, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses, Cryptosporidium, and Giardia) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%; C. perfringens, 61%; F-specific coliphages, approximately 40%; and enteric viruses, 31%. Cryptosporidium oocysts and Giardia cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable Cryptosporidium, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for Giardia cysts, Cryptosporidium oocysts, infectious Cryptosporidium, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study.

Potential use of a host associated molecular marker in Enterococcus faecium as an index of human fecal pollution.

Several genotypic and phenotypic microbial source tracking (MST) methods have been proposed and utilized to differentiate groups of microorganisms, usually indicator organisms, for the purpose of tracking sources of fecal pollution. Targeting of host-specific microorganisms is one of the approaches currently being tested. These methods are useful as they circumvent the need to isolate individual microorganisms and do not require the establishment of reference databases. Several studies have demonstrated that the presence and distribution of Enterococcus spp. in feces seems to be influenced by the host species. Here, we present a method for detection of genetic sequences in culturable enterococci capable of identifying human sources of fecal pollution in the environment. The human fecal pollution marker designed in this study targets a putative virulence factor, the enterococcal surface protein (esp), in Enterococcus faecium. This gene was detected in 97% of sewage and septic samples but was not detected in any livestock waste lagoons or in bird or animal fecal samples. Epidemiological studies in recreational and groundwaters have shown enterococci to be useful indicators of public health risk for gastroenteritis. By identifying the presence of human fecal pollution, and therefore the possible presence of human enteric pathogens, this marker allows for further resolution of the source of this risk.

Comparison of seven protocols to identify fecal contamination sources using Escherichia coli.

Microbial source tracking (MST) uses various approaches to classify fecal-indicator microorganisms to source hosts. Reproducibility, accuracy, and robustness of seven phenotypic and genotypic MST protocols were evaluated by use of Escherichia coli from an eight-host library of known-source isolates and a separate, blinded challenge library. In reproducibility tests, measuring each protocol's ability to reclassify blinded replicates, only one (pulsed-field gel electrophoresis; PFGE) correctly classified all test replicates to host species; three protocols classified 48-62% correctly, and the remaining three classified fewer than 25% correctly. In accuracy tests, measuring each protocol's ability to correctly classify new isolates, ribotyping with EcoRI and PvuII approached 100% correctclassification but only 6% of isolates were classified; four of the other six protocols (antibiotic resistance analysis, PFGE, and two repetitive-element PCR protocols) achieved better than random accuracy rates when 30-100% of challenge isolates were classified. In robustness tests, measuring each protocol's ability to recognize isolates from nonlibrary

Presence, infectivity, and stability of enteric viruses in seawater: relationship to marine water quality in the Florida Keys.

Concerns about the presence of enteric viruses in the surface waters of the Florida Keys prompted analyses of virus stability and persistence in these waters. In an in vitro study we evaluated the survival of poliovirus and stability of viral RNA in filtered natural seawater (FSW), unfiltered natural seawater (USW), artificial seawater (ASW) and DI water. This study compared cell culture infectivity with direct reverse transcription-polymerase chain reaction analysis. Attenuated poliovirus was seeded in the above water types and incubated in the dark at 22 and 30 degrees C for 60 days. At 22 degrees C, enhanced poliovirus survival and enhanced detection of viral RNA was observed in the seeded DI water control, artificial seawater and FSW samples. Detection of viruses in unfiltered seawater decreased rapidly at both temperatures by both methods of detection, suggesting that in the natural environment detection of enteroviral RNA may indicate a recent contamination event. In addition, in situ sampling in the Florida Keys during the late winter of 2000 revealed the presence of infectious enteroviruses at two sites and no sites exceeded recommended levels of microbial water quality indicators (enterococci or fecal coliform bacteria).

Reduction of poliovirus 1, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 on strawberries by physical and disinfectant washes.

The efficacy levels of different physical and chemical washing treatments in the reduction of viral and bacterial pathogens from inoculated strawberries were evaluated. Escherichia coli O157:H7, Salmonella Montevideo, poliovirus 1, and the bacteriophages PRD1, phiX174, and MS2 were used as model and surrogate organisms. Chemicals readily available to producers and/or consumers were evaluated as antimicrobial additives for the production of washes. The gentle agitation of contaminated strawberries in water for 2 min led to reductions in microbial populations ranging from 41 to 79% and from 62 to 90% at water temperatures of 22 and 43 degrees C, respectively. Significant reductions (> 98%) in numbers of bacteria and viruses were obtained with sodium hypochlorite (50 to 300 ppm of free chlorine), Oxine or Carnebon (200 ppm of product generating "stabilized chlorine dioxide"), Tsunami (100 ppm of peroxyacetic acid), and Alcide (100 or 200 ppm of acidified sodium chlorite) washes. Overall, 200 ppm of acidified sodium chlorite produced the greatest reductions of microorganisms. Hydrogen peroxide (0.5%) was slightly less effective than free chlorine in a strawberry wash and caused slight fruit discoloration. Cetylpyridinium chloride (0.1%) was effective in the reduction of bacterial species, while trisodium phosphate (1%) was effective against viruses. The consumer-oriented produce wash Fit was very effective (> 99%) in reducing the numbers of bacteria but not in reducing the numbers of viruses. Another wash, Healthy Harvest, was significantly less effective than Fit in reducing bacterial pathogens but more effective for viruses. The performance of automatic dishwashing detergent was similar to that of Healthy Harvest and significantly better than that of liquid dishwashing detergent. Solutions containing table salt (2% NaCl) or vinegar (10%) reduced the numbers of bacteria by about 90%, whereas only the vinegar wash reduced the numbers of viruses significantly (ca. 95%).

Geographical variation in ribotype profiles of Escherichia coli isolates from humans, swine, poultry, beef, and dairy cattle in Florida.

Waters impacted by fecal pollution can exact high risks to human health and can result in financial losses due to closures of water systems used for recreation and for harvesting seafood. Identifying the sources of fecal pollution in water is paramount in assessing the potential human health risks involved as well as in assessing necessary remedial action. Recently, various researchers have used the ribotyping method to identify sources of bacterial indicators (Escherichia coli and enterococci) in environmental waters. While these studies have identified genotypic differences between human- and animal-derived indicators that are capable of differentiating organisms isolated from humans and various animal hosts, most have focused on organisms collected from a confined geographic area and have not addressed the question of whether these ribotype profiles are watershed specific or if they can be applied universally to organisms from other geographic locations. In this study, E. coli isolates were obtained from humans, beef cattle, dairy cattle, swine, and poultry from locations in northern, central, and southern Florida and were subjected to ribotyping analysis. The intent was to determine (i) if ribotype profiles are capable of discriminating the source of E. coli at the host species level and (ii) if the resulting fingerprints are uniform over an extended geographic area or if they can be applied only to a specific watershed. Our research indicated that, using a single restriction enzyme (HindIII), the ribotyping procedure is not capable of differentiating E. coli isolates from the different animal species sampled in this study. Results indicate, however, that this procedure can still be used effectively to differentiate E. coli as being either human or animal derived when applied to organisms isolated from a large geographic region.

Preliminary evidence for human fecal contamination in corals of the Florida Keys, USA.

Corals and reef environments are under increased stress from anthropogenic activities, particularly those in the vicinity of heavily populated areas such as the Florida Keys. The potential adverse impacts of wastewater can affect both the environment and human health; however, because of the high decay rate of bacterial indicators in coral reef waters it has been difficult to document the presence of microbial contaminants and to assign risks in these environments. Here we show initial evidence that microorganisms associated with human feces are concentrated along the surface of coral heads relative to the overlying water column in the Florida Keys. Bacterial indicators (fecal coliform bacteria, enterococci or Clostridium perfringens) were detected in 66.7% of the coral surface microlayer (CSM) samples at levels between five and 1000 CFU/100 ml, but were found infrequently and at low numbers in the overlying water column ( < or = 2.5 CFU/100 ml). Similarly, enterovirus nucleic acid sequences, an indicator of human-specific waste, were detected in 93.3% of the CSM samples and only once in the water column by cell culture. Results show that coral mucus may accumulate enteric microorganisms in reef environments, and may indicate a risk to public and environmental health despite low indicator levels in the surrounding water.

Improved method for recovery of bacteriophage from large volumes of water using negatively charged microporous filters.

Current virus-recovery procedures using negatively charged microporous filters provide an inexpensive, reliable method for the recovery and detection of enteroviruses from water and wastewater; however, adjustment of the test samples to pH 3.5 to promote enterovirus adsorption results in significant inactivation of bacteriophage and an inability to simultaneously recover them from large volumes of water using this procedure. Procedures specifically designed for the detection of bacteriophage are currently in use but generally are only effective for small volumes of water. Positively charged filters can be used to recover both enteroviruses and bacteriophage from large volumes of water at neutral pH; however, the filters are expensive. The addition of manganese chloride to test solutions at pH 3.5 prior to filtration through negatively charged Filterite filters allowed for sampling of larger volumes of water by reducing the inactivation of bacteriophage and increasing the recovery of PRD1, MS2, and naturally isolated bacteriophage by a factor of four or five when compared with recoveries from solutions without MnCl2. This method provides an inexpensive, reliable alternative to large-volume bacteriophage recovery procedures that use positively charged filters at neutral pH.